期刊文献+
任意字段
题名或关键词
题名
关键词
文摘
作者
第一作者
机构
刊名
分类号
参考文献
作者简介
基金资助
栏目信息

转染乙酰胆碱结合蛋白基因到昆虫细胞的研究 被引量：1

Research of Transfection Acetylcholine Binding Protein cDNA into Insect Cells
在线阅读 下载PDF
导出
摘要 海兔(Aplysia californica)乙酰胆碱结合蛋白(Ac-AChBP)是研究烟碱型乙酰胆碱受体(nAChRs)的结构模板之一,它的高效表达对研究神经性相关疾病具有重要的意义。高效表达关键是要有高的转染效率,通过构建含(Ac-AChBP)基因的昆虫杆状病毒表达载体Bcmid,对脂质体转染Bcmid的多个条件进行探索,研究发现,当采用对数生长中期的细胞、细胞密度为2.5×106cells/mL,质粒DNA浓度为4.5×10-2g/mL和脂质体浓度为0.8 mL/L时,转染效率达到较高值,滴度为2×107±10%pfu/mL,同时证明高滴度病毒具有高的表达效率,而且表达蛋白具有生物活性。 Acetylcholine binding protein from Aplysia californica （Ac-AChBP） can provide a structural basis for understanding the structure and function of nicotinic acetylcholine receptors （nAChRs）. Its high level expres- sion is important to study neuropathic diseases, and high level expression need highly effective transfection. Bacmid containing Ac-A ChBP eDNA was constructed, and transfected insect cells through lipid. It was found to reach a high-titer （2×10^7±10% pfu/mL） at cell density of 2.5×10^6 cells/mL, recombinant Bacmid DNA con- centration 4.5×10^-6 g/mL, and lipid CellfeetinR II concentration 0.8 mL/L. It was also showed that high-titer virus had high level expression and the expressed recombinant Ac-AChBP had bioaetivity.
作者 林波 孟海玲 吴勇 邴晖 长孙东亭 罗素兰
机构地区 海南大学热带生物资源教育部重点实验室
出处 《生命科学研究》 CAS CSCD 北大核心 2014年第1期1-5,38,共6页 Life Science Research
基金 国家自然科学基金(41366002)资助项目 国家国际科技合作专项(2011DFR31210)资助项目 海南省社会发展科技专项(SF201328)资助项目 海口市重点科技计划(2013-16)资助项目 长江学者和创新团队发展计划(IRT1123)资助项目 海南省研究生创新科研课题
关键词 海兔乙酰胆碱结合蛋白(Ac-AChBP)基因 载体构建 脂质体转染 转染效率 表达效率 活性检测 acetylcholine binding protein eDNA from Aplysia californica （Ac-AChBP） vector construction lipid transfection transfection effciency expression efficiency bioactivity test
分类号 Q31 [生物学—遗传学]
  • 相关文献

参考文献16

  • 1KASHEVEROV I E, UTKIN Y N, TSETLIN V I. Naturally oc- curring and synthetic peptides acting on nicotinic acetylcholine receptors[J]. Current Pharmaceutical Design, 2009, 15(21): 2430- 2452.
  • 2BREJC K, SMIT A B, SIXMA T K, et al. Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors[J]. Nature, 2001, 411(6835): 269-276.
  • 3UNWIN N. Refined structure of the nicotinic acetylcholine re- ceptor at 4 A resolution[J]. Journal of Molecular Biology, 2005, 346(4): 967-989.
  • 4HAGA K, KRUSE A C, ASADA H, et al. Structure of the human M2 muscarinic acetylcholine receptor bound to an an- tagonist[J]. Nature, 2012, 482(7386): 547-551.
  • 5LEWIS R J, DUTERTRE S, VE33"ER I, et ol. Conus venom peptide pharmacology[J]. Pharmacological Reviews, 64(2):259- 298.
  • 6GEITMANN M, RETRA K, SMIT A B, et ol. Interaction kinet- ic and structural dynamic analysis of ligand binding to acetyl- choline-binding protein[J]. Biochemistry, 2010, 49(37):8143- 8154.
  • 7BUCHAPUDI K, XU X, ATAIAN Y, et al. Micromechanical measurement of AChBP binding for label-free drug discovery[J]. Analyst, 2011, 137(1):263-268.
  • 8HITCHMAN R B, POSSEE, R D, KING L A. Baculovirus ex- pression systems for recombinant protein production in insect cells[J]. Recent Patents on Biotechnology, 2009, 3 (1): 46-54.
  • 9WANG D, ZHANG S, LI, WANG X, et al. Structural insights into the assembly and activation of IL-113 with its receptors[J]. Nature Immunology, 2010, 11 (10): 905-911.
  • 10RICHARD B, HITCHMAN, ROBERT D. Baculovirus expres- sion systems for recombinant protein production in insect cells[J]. Recent Patents on Biotechnology, 2009, 3(1):46-54.

二级参考文献27

  • 1Bermingham A, Chand MA, Brown CS, et al. Severe respira- tory illness caused by a novel coronavirus, in a patient trans- ferred to the United Kingdom from the Middle East, Septem- ber 2012. Euro Surveil12012; 17:20290.
  • 2Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med 2012; 367:1814- 1820.
  • 3de Groot R J, Baker SC, Baric RS, et al. Middle East Respira- tory Syndrome Coronavirus (MERS-CoV); Announcement of the Coronavirus Study Group. J Viro12013; 87:7790-7792.
  • 4WHO. World Health Organization Global alert and response; Coronavirus infections. Available from: http://www.who.int/ csr/disease/coronavirus infections/en/.
  • 5Chan JF, Li KS, To KK, Cheng VC, Chen H, Yuen KY. Is the discovery of the novel human betacoronavirus 2c EMC/2012 (HCoV-EMC) the beginning of another SARS-like pandemic? J Infect 2012; 65:477-489.
  • 6Memish ZA, Zumla AI, A1-Hakeem RF, A1-Rabeeah AA, Stephens GM. Family cluster of Middle East respiratory syn- drome coronavirus infections. N Engl J Med 2013; 368:2487- 2494.
  • 7van Boheemen S, de Graaf M, Lauber C, et al. Genomic char- acterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans. MBio 2012; 3:e00473-e00412.
  • 8Woo PCY, Lau SKP, Li KSM, Tsang AKL, Yuen K-Y. Genetic relatedness of the novel human group C betaeoronavirus to Tylonycteris bat coronavirus HKU4 and Pipistrellus bat coro-navirus HKU5. Emerg Microbes Infect 2012; 1 :e35.
  • 9Raj VS, Mou H, Smits SL, et al. Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. Nature 2013; 495:251-254.
  • 10Jiang S, Lu L, Du L, Debnath AK. A predicted receptor-bind- ing and critical neutralizing domain in S protein of the novel human coronavirus HCoV-EMC. Jlnfect 2013; 66:464-466.

共引文献33

同被引文献2

引证文献1

二级引证文献1

生命科学研究
2014年 第1期

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部